Lesley Scott

Changes in Collagen Fibril Diameters Across Artery Walls Including a Correlation with Glycosaminoglycan Content

Collagen fibril diameters were measured at regular intervals across the walls of numerous arteries from human, pig and rat. In all vessels the smallest fibrils (mean-fibril diameters of 30-40 nm) occurred in the intima and inner media and the largest fibrils (MFDs 50-100 nm) in the outer adventitia. Between these two regions fibrils progressively increased in size. Circumferential and axial fibrils were of similar size and showed similar patterns of increase. At each sample site there was a range of diameters and frequency distributions were often multimodal with peaks 8 nm, or multiples of 8 nm, apart. Amounts of total and individual glycosaminoglycans (GAG) were determined at regular intervals across the wall of pig aorta and total and sulphated GAG levels were also determined at intervals across rat carotid artery using autoradiographic detection of incorporated [3H] glucosamine and 35S. In both vessels there was a strong correlation between decreasing GAG and increasing MFDs and over a narrow MFD range of 40 to 60 nm. These results demonstrate that collagen fibril diameters are excellent indicators to GAG levels and may be useful for making predictions about GAG levels in areas too small to sample biochemically.

Interaction of aortic endothelial and smooth muscle cells in culture Effect on glycosaminoglycan levels

Co-cultivation of various intra- and interspecific combinations of pig and rat aortic endothelial cells (AEC) and smooth muscle cells (SMC) resulted in a marked increase in hyaluronic acid (HA) levels, a smaller but significant increase in sulphated glycosaminoglycans (GAG), and an increase in the HA/sulphated GAG ratio, compared with the separate culture of the two cell types. Culture of SMC in AEC-conditioned medium produced similar changes in GAG levels whereas SMC-conditioned medium had no effect on AEC GAG levels. These results add further support for the concept that epithelial cells in general can modulate the GAG composition of adjacent connective tissue and thereby influence its morphological and physiological properties. It is suggested that the normal amounts, types and distribution of GAG in the arterial wall, and especially in the intima, may be partly dependent on interaction between the endothelium and SMC. It is further suggested that injury to endothelium, with a consequent failure in this interaction, could lead directly to changes in intimal GAG composition that contribute to lesion development.

Comparison of deposits of versican, biglycan and decorin in saphenous vein and internal thoracic, radial and coronary arteries: correlation to patency.

BackgroundMatrix proteoglycans versican, biglycan and decorin are important determinants of vessel‐wall structure and pathology. Thickened myxoid intimas typical of restenosis and early atherosclerosis are enriched in versican and biglycan, proteoglycans that promote proliferation and migration of smooth muscle cells and bind lipoproteins. In contrast, compact fibrous intimas are characterized by decorin. ObjectiveTo compare the distribution patterns of these matrix proteoglycans, and changes induced by organ culture in coronary artery, saphenous vein, internal thoracic artery (ITA), and radial artery, and correlate differences to patency. MethodsVessels were collected at the time of bypass surgery and heart transplantation and either fixed for immunohistochemistry or prepared for organ culture. Vessels in culture were labelled with [3H]‐glucosamine and processed for autoradiography and immunohistochemistry. Distribution patterns for proteoglycans and radio‐labelling were determined morphometrically. ResultsDistribution profiles in coronary artery and saphenous vein were similar, with relatively high levels of subendothelial versican and biglycan and low levels of decorin. In culture subendothelial incorporation of [3H]‐glucosamine and immunostaining for versican and biglycan, but not decorin, were significantly increased. In contrast, the thin intima of the ITA was relatively enriched in decorin compared with the medial layers and in culture intimal staining for decorin increased markedly compared with a modest increase for biglycan and no change for versican. There was an even distribution in radial artery of all three proteoglycans across the intima without subendothelial accumulations. In culture there was an increase in staining intensity for proteoglycans of the radial artery. Neither the ITA nor radial artery exhibited an increase in subendothelial incorporation of [3H]‐glucosamine in culture. ConclusionsThe distributions of proteoglycans, and responses to culture correlate to the known differences in patency between grafted saphenous vein and ITA and predict that the radial artery will outperform the saphenous vein but might not be as good as the ITA for long‐term patency.

Effect of TGF-β1 Antisense S-Oligonucleotide on Synthesis and Accumulation of Matrix Proteoglycans in Balloon Catheter-Injured Neointima of Rabbit Carotid Arteries

Arterial matrix proteoglycans (PG) are necessary for the maintenance of viscoelastic properties of the vessel wall, but excess levels, particularly of versican and biglycan in primary and restenotic intimal thickenings, are correlated with increased tissue volume and with atherogenicity. There is good evidence that the primary stimulus to increased PG synthesis, including versican and biglycan, is transforming growth factor-β1 (TGF-β1). The aim of this study was to determine the effects of reducing endogenous TGF-β1 on rates and patterns of PG synthesis and on versican, biglycan and decorin accumulation in vivo. Rabbit common carotid arteries subjected to balloon catheter injury were treated with a TGF-β1 antisense phosphorothioate oligonucleotide applied in a pluronic gel to the adventitia. Control animals received a nonsense oligonucleotide or gel alone. TGF-β1 antisense (1) significantly (p < 0.005) inhibited, at day 2, the balloon catheter-induced increase in TGF-β1 mRNA relative to β-actin mRNA; (2) inhibited intimal thickening at 23 days by ∼40% (p < 0.05); (3) inhibited (p < 0.05) PG synthesis, measured by autoradiographic detection of [3H]glucosamine, in the media of day 2 ballooned carotids and in the subendothelial zone of day 23 neointima, and (4) decreased immunostaining intensity for versican (p < 0.03) and TGF-β1 (p < 0.001) in the neointima. Biglycan was reduced to a lesser extent but not significantly and decorin was not affected. Proliferating cell nuclear antigen indices were variable and not significantly changed. These findings confirm a role for TGF-β1 in developing neointima and demonstrate a specific effect on the synthesis, distribution, and accumulation of matrix PG, particularly versican.

Culture of rat and pig aortic endothelial cells Differences in their isolation, growth rate and glycosaminoglycan synthesis

Abstract Aortic endothelial cells of the rat, an animal not normally predisposed to atherosclerosis, were found to resist removal from the vessel wall by collagenase digestion, whereas endothelial cells of the pig, which is susceptible to atherosclerosis, were readily removed by the same treatment. Under the same tissue culture conditions, rat endothelial cells, obtained from explants, had a faster rate of growth and could be passaged many more times than pig endothelial cells. Furthermore, in culture, rat endothelial cells produced large amounts of glycosaminoglycans (GAG), mostly (>80%) hyaluronic acid, while pig endothelial cells produced more moderate amounts which were mostly (>70%) sulphated. While it is not known whether these differences accurately reflect in vivo characteristics of rat and pig endothelium, it is suggested that species variability in the strength of attachment of endothelium to the underlying intima, in the rate of regeneration after damage, and in possible contribution to the GAG composition of the intimal matrix, may be correlated with susceptibility to atherosclerosis.

Interaction of epithelial cells and fibroblasts in culture: effect on glycosaminoglycan levels.

Abstract Twelve- to fifteen-day chick embryo liver cells (epithelial) were cultured on top of confluent chick embryo fibroblasts to produce an in vitro model of an epithelial-mesenchymal interacting system. This cocultivation resulted in a marked increase in hyaluronic acid (HA) levels and a decrease in chondroitin sulfate (CS) levels, either in total or in proportion to HA, compared with the two cell types cultured separately. The liver cells cultured alone produced little or no detectable glycosaminoglycans (GAGs). Cocultivation of increasing numbers of liver cells with fibroblasts resulted in a progressive increase and decrease of HA and CS levels, respectively, and the combined effect of these changes was a progressive increase in the HA/CS ratio. Fibroblasts cultured in liver-cell-conditioned growth medium also showed increased levels of HA, but in contrast to cocultivation, an increase in CS and no change in the HA/CS ratio. Liver cells cultured in fibroblast-conditioned growth medium showed no changes in GAG level. This suggests that under conditions of cocultivation the fibroblasts alone could be responsible for the increased HA levels and that the decreased CS levels are a result of conditions produced by the close proximity of the two cell types. In vivo most connective tissues immediately adjacent to epithelial tissues are also characterized by a matrix rich in HA and these results support the concept that some epithelial tissues are able to modulate the GAG composition of adjacent connective tissues and thereby affect their immediate extracellular environment.

ORGAN CULTURE OF RAT CAROTID ARTERY: MAINTENANCE OF MORPHOLOGICAL CHARACTERISTICS AND OF PATTERN OF MATRIX SYNTHESIS

SummarySegments of rat carotid artery were maintained in serum-free or serum-supplemented medium for 2 wk, and at intervals of 3, 7, and 14 d the morphology and pattern of matrix synthesis were compared to those in vivo. In serum-free medium and 0.2% serum both the endothelium and the smooth muscle cells (SMC) could be maintained with a minimum of change for 7 d and without substantial change for 14 d. In 2% and 10% serum there was little change for the first 3 d, but subsequently there was a progressive overlapping of the endothelial cells to produce a 3 to 4 layered cell sheet, often separated from the subendothelial matrix; the SMC, however, did not appear to proliferate or migrate and in general retained their typical cellular features for the full time in culture. The synthesis of matrix components was measured by autoradiographic detection of incorporated [3H]glucosamine and35S. At all time periods and serum concentrations the percentage distribution for each label across the arterial wall was found to be similar to that in live animals injected with the same labels. [3H]Glucosamine predominated in the endothelium and the narrow subendothelial layer, which together make up the intima, whereas35S predominated in the media. In vitro more than 50% of the [3H]glucosamine in the intima and 40% in the adjacent first layer of the media was susceptible toStreptomyces hyaluronidase. As the morphology of both cell types and their synthesis of matrix components could be maintained in organ culture without substantial change we believe that the rat carotid artery may be a suitable model for the investigation of factors affecting arterial structure.

Modification by Suppressor Cells and Serum Factors of the Cell-Mediated Immune Response in Experimental Pyelonephritis

A marked suppression of the thymusderived (T)-lymphocyte response to concanavalin A has been demonstrated in vitro during renal infection. Suppression of the T-lymphocyte response in vitro was seen as early as 2 h after the induction of renal infection, but maximum suppression was found 24-72 h later. A population of suppressor cells in the splenic lymphocyte population, generated during the hosts response to infection, contributed to the depressed lymphocyte response. Removal of suppressor cells restored the mitogenic responsiveness of the remaining splenic lymphocytes. Conversely, in co-culture experiments, a suppressor cell present in the splenic lymphocyte population of pyelonephritic animals was shown to be capable of suppressing the mitogenic responsiveness of normal splenic lymphocytes. Significantly reduced host vs. graft responses by the pyelonephritic animals confirmed, in vivo, the depression of cell-mediated immune mechanisms. An additional suppressive factor was found in the serum of pyelonephritic animals which depressed in vitro the mitogenic responsiveness of splenic lymphocytes from normal animals. Support for the suppressive role of this serum factor was found when splenic lymphocytes from pyelonephritic animals were tested in vivo in the absence of homolgous serum (graft vs. host). Under these conditions, the lymphocytes showed an enhanced reaction compared with lymphocytes from normal animals. The presence of a suppressor cell population and a serum factor, both capable of depressing cell-mediated mechanisms, may be major factors contributing to the establishment of infection in the kidney.

Endothelial cell stimulation of smooth muscle glycosaminoglycan synthesis can be accounted for by transforming growth factor beta activity

Endothelial cell conditioned medium (ECCM) contains a factor which markedly stimulates smooth muscle cell (SMC) glycosaminoglycan (GAG) synthesis. We report here that the factor responsible is transforming growth factor beta (TGF-beta) as assessed by (1) protease and thiol sensitivity, (2) heat and acid enhancement of ECCM activity, and (3) neutralisation of ECCM activity by anti-TGF-beta-immunoglobulin. Anti-TGF-beta-neutralisation was effective against increases in both sulphated and non-sulphated GAG. Previous studies showed that ECCM from EC of varying densities stimulated individual GAG to varying degrees. ECCM from low density EC preferentially stimulated hyaluronic acid (HA) whereas ECCM from intermediate and high density cultures stimulated increasing amounts of sulphated GAG. Exposure of SMC to varying concentrations of TGF-beta produced a similar pattern Exposure of SMC to varying concentrations of TGF-beta produced a similar pattern of response. Very low amounts of TGF-beta (less than 10-500 pg/10 cells) stimulated a marked and significant increase in HA synthesis. Increase in chondroitin sulphate 4/6 was most marked at TGF-beta levels from 500-1000 pg/10(6) cells. At levels above 1000 pg/10(6) cells both HA and sulphated GAG synthesis decreased but still remained elevated above controls. These findings indicate that TGF-beta alone can account for the changes in SMC GAG synthesis stimulated by ECCM. It was also found, however, that heat-treated SMC conditioned medium stimulated SMC GAG synthesis, thus SMC may contribute to the control of their own GAG synthesis through autocrine TGF-beta activity.

Subendothelial proteoglycan synthesis and transforming growth factor beta distribution correlate with susceptibility to atherosclerosis.

Coronary bypass vessels, saphenous vein (SV) and internal thoracic artery (ITA), differ in susceptibility to atherosclerosis and medium- to long-term patency. Whereas most ITA remain patent (90% at 10 years), 20% of SV grafts fail in the first year and approximately 45% fail within 10 years. Reasons for these differences are not fully understood. Loss of SV patency may reflect early metabolic events, particularly increased proteoglycan (PG) synthesis which contributes to intimal volume and promotes atherogenesis through retention of atherogenic lipoproteins. We determined, in vitro, the PG metabolic activity of SV, ITA, and human coronary arteries through autoradiographic detection of incorporated [3H]glucosamine. SV had significantly higher levels of PG synthesis than ITA, especially in the subendothelial zone and after time (7 days) in culture. Patterns of synthesis in coronary vessels were similar to SV with high levels of incorporation in the subendothelial zone of thickened intima (> 100 microm). Increased subendothelial labelling in SV was due to increased PG synthesis, not decreased degradation. ITA showed no propensity for upregulation of subendothelial PG synthesis. Immunohistochemistry showed TGF-beta1 and TGF-beta2 localised primarily to the subendothelial zone of SV and coronary arteries. With time in culture immunostaining increased in parallel with increased PG synthesis. Subendothelial TGF-beta1 and TGF-beta2 were absent in ITA. A panspecific TGF-beta neutralising antibody reduced subendothelial PG synthesis in SV and coronary arteries by 50 and 60%, respectively. These results support the idea that vessels susceptible to atherosclerosis show increased accumulation of subendothelial PG mediated by TGF-beta.